Flex-nucleoside analogues, novel therapeutics against  filoviruses and flaviviruses

ABSTRACT

The present invention is directed to compounds, methods and compositions for treating or preventing viral infections using nucleosides analogs. Specifically, the present invention provides for the design and synthesis of acyclic fleximer nucleoside analogues having increased flexibility and ability to alter their conformation structures to provide increased antiviral activity potential with the result of inhibiting flaviviruses, filoviruses and/or coronaviruses.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.:62/539,034 filed on Jul. 31, 2017, the contents of which is incorporatedby reference herein for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Number GrantNumber R21AI097685 and T32GM066706 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF INVENTION

The present invention is directed to compounds, methods and compositionsfor treating or preventing filoviruses and/or flaviviruses usingnucleosides analogues. Specifically, the present invention provides forthe design and synthesis of acyclic fleximer nucleoside analogues havingincreased flexibility and ability to alter their conformation to provideincreased antiviral activity potential with the result of inhibitingseveral coronaviruses.

BACKGROUND OF THE INVENTION

Viruses are small infectious agents that can only multiply within thecells of animals, plants, and bacteria. The structures of viruses aresimple compared to living cells and contain a small haploid DNA or RNAgenome and a protein or glycoprotein coat called a capsid. In addition,some viruses called enveloped viruses are surrounded by a lipidmembrane.

A number of viruses appear on the United States National Institutes ofAllergy and Infectious Disease (NIAID) list of EmergingDiseases/Pathogens list, which include Flaviviruses (Dengue, Zika andWest Nile) and Filoviruses (Ebola, Sudan and Marburg) to name a few.

Filoviruses are enveloped viruses with a genome consisting of one linearsingle-stranded RNA segment of negative polarity. The viral genomeencodes 7 proteins. Nucleoprotein (NP), virion protein 35 kDa (VP35) andvirion protein 30 kDa (VP30) are associated with the viralribonucleoprotein complex. Members of the filovirus genus include ZaireEbola virus, Sudan Ebola virus, Reston Ebola virus, Cote d'Ivoire Ebolavirus and Marburg virus. Ebola and Marburg viruses can cause severehemorrhagic fever and have a high mortality rate. Ebola virus (Zaire andSudan species) was first described in 1976 after outbreaks of a febrile,rapidly fatal hemorrhagic illness were reported along the Ebola River inZaire (now the Democratic Republic of the Congo) and Sudan. The naturalhost for Ebola viruses is still unknown. Marburg virus, named after theGerman town where it was first reported in 1967, is primarily found inequatorial Africa. The host range of Marburg virus includes non-humanand human primates. Marburg made its first appearance in Zimbabwe in1975 and was later identified in other African countries, includingKenya (1980 & 1987) and Democratic Republic of the Congo (1999).

Viruses in the genus flavivirus are known to cause viral hemorrhagicfevers (VHFs). Flaviviruses are enveloped viruses with a genomeconsisting of one linear single-stranded RNA segment of positive polaThe polyprotein is co- and post-transcriptionally cleaved by cell signalpeptidase and the viral protease to generate individual viral proteins.Viral structural proteins include capsid (C), precursor to M (prM),minor envelope (M) and major envelope (E).

Members of the flavivirus genus include yellow fever virus, Apoi virus,Aroa virus, Bagaza virus, Banzi virus, Bouboui virus, Bukalasa batvirus, Cacipacore virus, Carey Island virus, Cowbone Ridge virus, Dakarbat virus, dengue virus, Edge Hill virus, Entebbe bat virus, GadgetsGully virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus,Japanese encephalitis virus, Jugra virus, Jutiapa virus, Kadam virus,Kedougou virus, Kokobera virus, Koutango virus, Kyasanur Forest diseasevirus, Langat virus, Louping ill virus, Meaban virus, Modoc virus,Montana myotis leukoencephalitis virus, Murray Valley encephalitisvirus, Ntaya virus, Omsk hemorrhagic fever virus, Phnom Phenh bat virus,Powassan virus, Rio Bravo virus, Royal Farm virus, Saboya virus, SalVieja virus, San Perlita virus, Saumarez Reef virus, Sepik virus, St.Louis encephalitis virus, Tembusu virus, tick-borne encephalitis virus,Tyuleniy virus, Uganda S virus, Usutu virus, Wesselsbron virus, WestNile virus, Yaounde virus, Yokose virus, Zika virus, cell fusing agentvirus and Tamana bat virus.

There are relatively few prophylactic or therapeutic agents fortreatment of viral diseases caused by Flaviviruses and Filoviruses. Theneed for new and more effective antiviral therapeutics, particularlythose targeting emerging and reemerging infectious diseases andpathogens continues to increase. Thus, in light of the above discussion,there is a need for discovering and providing new and more efficientantiviral drugs.

SUMMARY OF THE INVENTION

The present invention provides for flexible and modified nucleosideanalogues that allow access to more potential binding sites with theability to retain their potency against viral diseases caused byFlaviviruses and Filoviruses since they can “wiggle and jiggle” in thebinding site. These findings are causing a paradigm shift in drug designhaving antiviral activity.

In one aspect, the present invention provides for a series of doublyflexible nucleoside analogues based on the acyclic nucleosides and theflex-base moiety found in the fleximers having antiviral activityagainst Flaviviruses and Filoviruses selected from compounds accordingto the following:

Wherein Ac is CH3-C(═O), or pharmaceutically acceptable salt, isomer,hydrate, prodrug or solvate thereof.

In another aspect, the present invention provides for the use ofmodified nucleosides of the present invention in a medicament formedicine. In a more specific embodiment hereof, said use as a medicineis for the prevention or treatment of a filovirus, flavivirus and/orcoronavirus in a subject, mammal or human. Preferably, a therapeuticallyeffective amount of the acyclic fleximer nucleoside analogue is from0.05 to 50 mg per kilogram body weight of the subject per day.

In yet another aspect, the present invention provides for contacting acell infected with a filovirus or flavivirus or to be infected with afilovirus or flavivirus with at least one of the modified nucleosidesprovided herein, wherein the amount of the modified nucleosides is fromabout 1 μg/ml to about 40 μg/ml, and more preferably, from about 3 μg/mlto about 20 μg/ml.

In another aspect, the present invention provides for the manufacture ofa medicament comprising the modified nucleosides of the presentinvention for the treatment of a filovirus or flavivirus.

In a further aspect, the present invention provides for the use of themodified nucleosides of the present invention for the prevention ortreatment of a filovirus and/or flavivirus, wherein the modifiednucleosides comprise the compounds 1, 2, 3 or 9.

In a still further aspect, the present invention provides for apharmaceutical composition comprising at least one of the modifiednucleosides of the present invention and a pharmaceutically acceptablecarrier.

In another aspect, the invention also provides novel intermediates orprodrugs which are useful for preparing the compounds of the inventionor converted to active agents in vivo, respectively. Prodrugs areselected and prepared in order to improve some selected property of themolecule, such as water solubility or ability to cross a membrane,temporarily. Most common (biologically labile) functional groupsutilized in prodrug design include carbonates, esters, amino acylesters, amides, carbamates, oximes, imines, ethers or phosphates.

In yet another aspect the present invention provides for nucleosideanalogues based on the acyclic nucleoside acyclovir (ACV) selected fromthe following compounds:

or a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.

In a further aspect, the invention also provides a method of inhibitinga filovirus or flavivirus administering to a mammal infected with such afilovirus or flavivirus a compound selected from compounds 1, 2, 3 or 9and pharmaceutically effective salts thereof in an amount to effectivelyinhibit the replication of a filovirus or flavivirus in infected cellsin the mammal.

In a still further aspect, the present application provides for a methodof treating a filovirus, flavivirus or coronavirus in a patient,comprising administering to said patient a therapeutically effectiveamount of a compound of the present invention, and at least oneadditional therapeutic agent having anti-viral properties.

In other aspects, methods for synthesis, analysis, separation,isolation, purification, characterization, and testing of the compoundsof this invention are provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of the target flexible nucleoside analoguesof the present invention.

FIG. 2 shows the dose response of Control compound E864 for treatment of(A) EBOV, (B) MARV and (C) SUDV.

FIG. 3 (A) shows the percentage inhibition (% INH) and FIG. 3 (B)percent viability for compounds HP105 and MR064 for inhibition of EBOVin Hela cells.

FIG. 4 (A) shows the percentage inhibition (% INH) and FIG. 4 (B)percent viability for compounds HP083, HP105 and MR064 for inhibition ofSUDV in Hela cells.

FIG. 5 (A) shows the percentage inhibition (% INH) and FIG. 5 (B)percent viability for compounds HP083, HP105 and MR064 for inhibition ofMARV in Hela cells.

FIG. 6 shows MTase activity for multiple viruses using sinefungin as acontrol and comparing to compounds 2MR04, MR064, HP105 and HP083.

FIG. 7 shows MTase activity for multiple viruses using sinefungin as acontrol and comparing to compounds 2MR04, MR064, HP105 and HP083.

FIG. 8 shows MTase activity for multiple viruses using the 2MR04compound.

FIG. 9 shows MTase activity for ZIKA virus using compounds 2MR04, MR064and HP105.

FIG. 10 shows MTase activity for Dengue virus using compounds 2MR04,MR064 and HP105.

FIG. 11 shows test results using different viral inhibitors, compared toNTPs as a control, in the testing of MERS.

FIG. 12 shows test results using different viral inhibitors, compared toNTPs as a control, in the testing of SARS-CoV.

FIG. 13 shows blots of formation of nucleotide sequences using the 2MR04inhibitor against MERS, SEQ ID NO 1.

FIG. 14 shows blots of formation of nucleotide sequences using the 2MR04inhibitor against SARS, SEQ ID NO. 1.

FIG. 15 shows the effect of 2MR04 on Zika Virus.

DETAILED DESCRIPTION OF THE INVENTION

Unique nucleoside analogues have been termed ‘fleximers’ and weredesigned to explore how nucleobase flexibility affects the recognition,binding, and activity of nucleoside(tide) analogues. The fleximerspossess a purine base scaffold in which the pyrimidine moiety isattached by a single carbon-carbon bond, rather than being ‘fused’ as istypical for the purines. These analogues are designed to retain all ofthe requisite purine hydrogen bonding patterns while allowing thenucleobase to explore alternative binding modes.

The present invention provides for a series of doubly flexiblenucleoside analogues based on the acyclic sugar scaffold of acyclovirand the flex-base moiety found in the fleximers. The target compoundswere evaluated for their antiviral potential and found to inhibitfiloviruses, flaviviruses or coronaviruses.

Mammal or human hosts infected with a filovirus, flavivirus orcoronavirus can be treated by administering to said mammal or human aneffective amount of an acyclic fleximer nucleoside analogue of thepresent invention and such compounds can be administered by anyappropriate route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid or solid form.

The present invention relates to a method for treating a filoviral,flaviviral or coronaviral infection, comprising the administration, to apatient, of an effective amount of at least one acyclic fleximernucleoside analogue of the present invention and/or of a compositioncontaining same. In general, the acyclic fleximer nucleoside analogues,as active agents, of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The effectiveamount will be that amount of an acyclic fleximer nucleoside analogue ofthe present invention that would be understood by one skilled in the artto provide therapeutic benefits. The active agent can be administeredonce a week, twice or more times per week, once a day, or more than oncea day. As indicated above, all of the factors to be considered indetermining the effective amount will be well within the skill of theattending clinician or other health care professional.

For example, therapeutically effective amounts of an acyclic fleximernucleoside analogue of the present invention may range fromapproximately 0.05 to 50 mg per kilogram body weight of the subject perday; preferably about 0.1-25 mg/kg/day, more preferably from about 0.5to 10 mg/kg/day. Thus, for administration to a 70 kg person, the dosagerange would most preferably be about 35-700 mg per day.

In general, an acyclic fleximer nucleoside analogue of the presentinvention can be administered as pharmaceutical compositions by any oneof the following routes: oral, systemic (e.g., transdermal, intranasalor by suppository), or parenteral (e.g., intramuscular, intravenous orsubcutaneous) administration. Compositions can take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions.

The choice of formulation depends on various factors such as the mode ofdrug administration and bioavailability of the acyclic fleximernucleoside analogue. For delivery via inhalation the compound can beformulated as liquid solution, suspensions, aerosol propellants or drypowder and loaded into a suitable dispenser for administration.

A composition comprising an acyclic fleximer nucleoside analogue of thepresent invention may be combined with at least one pharmaceuticallyacceptable carrier, excipient or diluent. Some examples of acceptableexcipients are those that are non-toxic, will aid administration, and donot adversely affect the therapeutic benefit of the compound of theinvention. Such excipient may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one of skill in the art.

Solid pharmaceutical excipients useful in the invention may includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. Preferred liquid carriers, particularly forinjectable solutions, include water, saline, aqueous dextrose, andglycols. Other suitable pharmaceutical excipients and their formulationsare described in Remington's Pharmaceutical Sciences, edited by E. W.Martin (Mack Publishing Company, 18th ed., 1990). The amount of anacyclic fleximer nucleoside analogue of the present invention can varywithin the full range employed by those skilled in the art. For example,a composition may contain, on a weight percent (wt %) basis, from about0.01-99.99 wt % of an acyclic fleximer nucleoside analogue of thepresent invention based on the total formulation, with the balance beingone or more suitable pharmaceutical excipients.

The pharmaceutical composition according to the invention preferablycomprises an amount of an acyclic fleximer nucleoside analogue of thepresent invention of between 5 μg and 1000 mg, preferably between 1 and500 mg, preferably between 5 and 100 mg. The ratio between the amountsby weight of an acyclic fleximer nucleoside analogue of the presentinvention and of pharmaceutically acceptable carrier is between 5/95 and95/5, preferably between 20/80 and 80/20.

The acyclic fleximer nucleoside analogues of the present invention maybe the only active ingredients, or they may be combined with otheractive ingredients. The pharmaceutical composition according to theinvention may thus also comprise at least one other pharmaceuticalactive agent, in particular at least one other medicament used for thetreatment of viral infection. In particular, the composition accordingto the invention may also comprise, or be combined with, one or moreother antivirals. Generally, any antiretroviral may be combined, namelynucleoside or nucleotide and non-nucleoside inhibitors, proteaseinhibitors, entry inhibitors, etc.

The acyclic fleximer nucleoside analogues of the present invention orcompositions comprising same may be administered in various ways and invarious forms. Thus, they may be administered systemically, orally, byinhalation or by injection, for instance intravenously, intramuscularly,subcutaneously, transdermally, intra-arterially, etc., intravenous,intramuscular, subcutaneous and oral administration. For the injections,the acyclic fleximer nucleoside analogues of the present invention aregenerally conditioned in the form of liquid suspensions, which can beinjected by means of syringes or infusions, for example. In this regard,the acyclic fleximer nucleoside analogues of the present invention aregenerally dissolved in buffered, isotonic, physiological, saline, etc.,solutions which are compatible with pharmaceutical use and known tothose skilled in the art. Thus, the compositions may contain one or moreagents or carriers chosen from dispersants, solubilizing agents,stabilizers, preservatives, etc. Agents or carriers which can be used inliquid and/or injectable formulations are, in particular,methylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.

The acyclic fleximer nucleoside analogues of the present invention canalso be administered in the form of gels, oils, tablets, suppositories,powders, gel capsules, capsules, aerosols, etc. For this type offormulation, an agent such as cellulose, carbonates or starches isadvantageously used.

Generally, for the purpose of the present invention, solvates ofpharmaceutically acceptable solvents such as water and ethanol areequivalent to those not in forms of solvates.

The present invention relates to a method for treating a viralinfection, comprising the administration, to a patient, of an effectiveamount of at least one acyclic fleximer nucleoside analogue of thepresent invention and/or of a composition containing same. The acyclicfleximer nucleoside analogue can further be prodrugs or in form ofcapable of releasing the active ingredient after in vivo metabolism.

Since the first reported fatal outbreak in the mid 1970s, members of theFiloviridae virus family, including the Ebola virus (EBOV), the Sudanvirus (SUDV), and the Marburg virus (MARV), have continued to devastatemany areas across the globe, with mortality rates as high as 90%.(1,2)One of the worst outbreaks of EBOV occurred in West Africa from 2013 to2016, with over 28,000 documented infections and claiming more than11,000 lives, including nearly 900 health care workers.(1) Filovirusesare a group of enveloped, single-stranded, negative-sense RNA virusesthat cause fatigue, vomiting, and severe hemorrhagic fevers.(1,3,4)Members of the Filoviridae family are zoonotic viruses, where theprimary reservoir is speculated to be fruit bats, however, it is unclearif this is the only reservoir or how the transmission to humansoccurs.(2) The filoviruses are highly contagious and can easily spreadthrough interaction with an infected individual by direct contact withbodily fluids including vomit, sweat, saliva, and respiratorysecretions.2,4 With the high potential for re-emergence of these lethalviruses, particularly due to “super-spreaders”,(5,6) It is imperativethat a viable treatment option be identified in order to better fightthese crippling pathogens before the next outbreak occurs.

While various therapeutic options have been pursued includingvaccines,(7) monoclonal antibodies,(4,8) and recombinant proteins,(9,10) many of these have yet to reach clinical trials and mayultimately not translate well to effective treatments that can be madereadily available during an outbreak, particularly in suboptimalconditions.(11)

The present invention provides a therapeutic option using the nucleosideanalogues as shown in FIG. 1. Nucleoside analogues have long been thecornerstone of antiviral therapies due to their ability to inhibit viralreplication because they mimic the structure of the naturalnucleosides.(12,13) As such, they can be recognized by cellular or viralenzymes, including the viral DNA or RNA polymerases. Moreover, becausethey contain various structural modifications, this leads to cessationof viral replication, typically due to chain termination.(13) Variousnucleoside analogues against filoviruses such as EBOV have already beenproposed including S-adenosylhomocysteine hydrolase (SAHase) inhibitorsc3Ado and c3Nep (14,15) and the monophosphate derivative of BCX4430,(16)an adenosine analogue that acts as a non-obligate chain terminator,however, none of these have progressed to the clinic. Most recentlyGS-5734, a monophosphoramidate prodrug adenosine analogue which targetsEBOV RNA-dependent RNA polymerase (RdRp), exhibited very potent activityagainst both EBOV and MARV,(17,18) further demonstrating the potentialfor finding effective nucleoside inhibitors of filoviruses. Over thepast several years, research by the present inventor has focused on thedevelopment of flexible nucleoside analogues, termed “Fleximers”.(19-26)

Herein, the anti-filovirus activity is reported for the analogues inFIG. 1, as well as the corresponding phosphoramidate prodrug 3.

The synthesis of the target compounds began with the substitutedimidazole 4, utilizing the routes previously employed in our group(Scheme 1).(26)

Treatment with sodium sulfite in a 30% ethanol/water solution resultedin simultaneous deacetylation and selective deiodination to provide keyintermediate 5. Acetylation of 5 then generated 6, the 5′ protectedintermediate needed for the prodrug synthesis. In parallel, theorganometallic coupling reagent 7 was synthesized starting from thecommercially available 2-amino-4-methoxypyrimidine.(27,28) Stillecoupling of 7 to 5 gave 1. Alternatively, using the acetylated 6, Stillecoupling provided the desired double prodrug 2.

Synthesis of the McGuigan ProTide (29-33) started with commerciallyavailable L-alanine and utilized literature procedures to generate thephosphoramidate 8 (Scheme 2).(34) Reaction of 8 with fleximer 1 in thepresence of tert-butyl magnesium chloride then provided the desiredMcGuigan ProTide 3 in 69% yield.

After the successful synthesis of the three Flex-analogues 1, 2, and 3,the compounds were screened against a panel of filoviruses includingEBOV, MARV, and SUDV, as well as other hemorrhagic fever viruses such asLassa and Rift Valley Fever. The first series of assays utilized HeLacells infected with live-virus isolates of EBOV (Makona), SUDV (Gulu),and MARV (Ci67). Activity against all three viruses was observed for theMcGuigan prodrug 3, with the best activity against SUDV (Table 1).

The second series of assays utilized Huh7 cells infected withrecombinant reporter EBOV, Lassa, and Rift Valley Fever viruses. Asobserved in the first series of assays, compound 3 was active againstEBOV at a similar concentration, however, compound 1 exhibited the bestactivity (EC₅₀=2.2±0.3 lM) against EBOV in Huh7 cells (Table 2).

TABLE 1 Antiviral activity of nucleoside analogues in infected HeLacells, values are in 1M. EBOV SUDV MARV CMPD EC₅₀ CC₅₀ EC₅₀ CC₅₀ EC₅₀CC₅₀ 1 >100 >100 >100 >100 >100 >100 2 44 ± 13 >100 20 ± 10 >100 70 ±27 >100 3 29 ± 9  >100 7 ± 2 >100 62 ± 13 >100 EC₅₀: Effectiveconcentration showing 50% inhibition of virus-induced Cytopathic effectCPE CC₅₀: Cytotoxic concentration showing 50% inhibition of cellsurvival

TABLE 2 Antiviral activity of nucleoside analogues against recombinantreporter viruses in Huh7 cells in 1M. EBOV Lassa Virus Rift Valley FeverCMPD EC₅₀ CC₅₀ EC₅₀ CC₅₀ EC₅₀ CC₅₀ 1 2.2 ± 0.3 >50 >50 >50 >50 >50 327.2 ± 2.2  >50 >50 >50 >50 >50 EC₅₀: Effective concentration showing50% inhibition of virus-induced Cytopathic effect CPE CC₅₀: Cytotoxicconcentration showing 50% inhibition of cell survival

Infectious diseases such as EBOV continue to pose a serious healththreat due to the high mortality rates associated with these deadlyviruses. While ongoing studies have identified various therapeutics aspotential EBOV treatments, there are limited vaccines or therapeuticsavailable, and as such, it is imperative that an effective treatmentoption is developed. Within this study it was found that both compounds1 and 3 exhibited antiviral activity against a recombinant reporter EBOVin Huh7 cells, though surprisingly the McGuigan prodrug was ˜10-foldless potent (EC₅₀=2.2±0.3 lM and 27.2±2.2 lM respectively). Againstwild-type viruses in HeLa cells, compound 1 had no detectable activity,though compound 3 inhibited both EBOV and SUDV (EC₅₀=29±9 and 7±2 lMrespectively). The difference in activity of 1 in the Huh7 cellscompared to the HeLa cells is most likely due to a difference inspecific metabolism of the compound in those cells lines.

Three compounds in Table 3 were tested in Ebola/Makona (except Hp083),Ebola/Sudan and Marburg infection assay with HeLa, and HFF cell lines.Stock solutions were made at 10 mM in 100% DMSO. Compounds weredispensed 2 h before infection by HP D300 directly from the 100% DMSOstock into assay wells with cells. DMSO was normalized in all wells tofinal 1%. Compound activity was tested in a 8-point dose response with 3fold step dilution (see table below). The titration started at 100 μM(final) and repeated 3 times on a single plate (n=3).

TABLE 3 1 2 3 4 5 6 7 8 HP105 100 33.33 11.11 3.70 1.23 0.41 0.14 0.05MR064 100 33.33 11.11 3.70 1.23 0.41 0.14 0.05 HP083 100 33.33 11.113.70 1.23 0.41 0.14 0.05

16 wells were treated with 1% DMSO to be used as a neutral control.Additionally, 16 wells were not infected and were used as a low signalcontrols. Cells were infected with EBOV(Makon) in Hela with MultiplicityOf Infection (MOI)=1.5, Marburg (Ci-67) MOI=1.0 and Sudan (Gulu)MOI=0.1. Infection was stopped after 48 h by fixing cells with aformalin solution.

To detect infected cells an immuno-staining was completed with anti-GPantibodies. Images were acquired by the PE Opera confocal platform usinga 10× objective and were analyzed using Acapella software. Signal forGP-staining was converted into % infection. The number of nuclei perwell was used to determine % viability of cells (in comparison toinfected but untreated controls). Data was analyzed using GeneDatasoftware. % of infection was converted into % Inhibition (%INH) for eachwell using plate controls.

FIG. 2 shows the plates statistics for the three different virusesindicating the level of infection and number of nuclei. Also includedare three graphs showing the use of a control compound E864 on the threedifferent viruses.

FIGS. 3, 4 and 5 show that one of the tested nucleosides, that beingnucleoside 3, demonstrated highest antiviral activity in Hela cellsagainst EBOV (EC₅₀ 29.10), SUDV (EC₅₀ 6.85) and MARV (EC₅₀ 62.29).

A number of flaviviruses cause human disease, particularly hemorrhagicfevers and encephalitis. Each species of flavivirus has a uniquegeographic distribution; however, taken together, flaviviruses, andflavivirus-induced disease, can be found world-wide. One of the morecommonly known diseases is dengue fever, or dengue hemorrhagicfever/shock, which was first described as a virus-induced illness in1960. Dengue fever occurs in tropical and temperate climates-and isspread by Aedes mosquitoes. The symptoms include febrile headache, jointpain, rash, capillary leakage, hemorrhage and shock. Another commonflavivirus-induced disease is yellow fever. Yellow fever is found intropical Africa and America and is transmitted by mosquitoes. Themortality rate is approximately 30% and symptoms include febrileheadache, myalgia (muscle pain), vomiting and jaundice. Examples of someof the other diseases caused by flavivirus species include Japaneseencephalitis, and West Nile fever. These diseases share many of the samesymptoms, which may include headache, myalgia, fever, hemorrhage,encephalitis, paralysis and rash.

Dengue virus (DV) is a mosquito-borne pathogen that causes dengue fever(DF) and severe life threatening illness, dengue hemorrhagicfever/dengue shock syndrome (DHF/DSS). DV is a small, enveloped,positive-stranded RNA virus that belongs to the Flavivirus genus of theFlaviviridae family which also includes Zika virus, and yellow fevervirus. Zika virus (ZIKV) is an arthropod-borne Flavivirus and since 2015has rapidly spread through South and Central America and the Caribbean.ZIKV was first discovered in Uganda in 1947 in the course of mosquitoand primate surveillance. The infection, known as Zika fever, oftencauses no or only mild symptoms, and had until now remained a raredisorder confined within a narrow equatorial belt from Africa to Asia.The World Health Organization (WHO) has declared the Zika virus aninternational public health emergency, prompted by growing concern thatan increased incidence of microcephaly in fetuses born to mothersinfected with ZIKV is linked to the recent outbreaks.

Additional testing was conducted on multiple types of viruses usingcompounds 2MR04, MR064, HP105 and HP083 and sinefungin as an inhibitorypositive control. The compounds were solubilized in water (1 mM) with afinal concentration of 50 μM. The following enzymes were used(independent triplicates, except for DV experiment performedinduplicate):

-   1) SARS-CoV nsp14 (N7 MTase): 10 nM-   2) MERS-CoV nsp14 (N7 MTase): 125 nM-   3) MERS-CoV nsp10/nsp16 (2′O MTase): 500 nM-   4) Zika virus NS5 MTase: 500 nM-   5) Dengue virus NS5 MTase: 500 nM-   6) hPMV CRIV+: 1 μM-   7) human N7 MTase (hN7 or RNMT): 20 nM

Methyltransferase (MTase) is involved in RNA capping process andimportant for mRNA stability, protein synthesis and also virusreplication, thus MTase is an attractive target in designing MTaseinhibitors. Sinefungin, a well-known anti-fungal AdoMet analogue andwhich is known to inhibit methylation and viral replication in otherflaviviruses, thus, making MTase a valid target anti-virus drugdiscovery

The results are shown in FIG. 6 for severe acute respiratory syndrome(SARS), Middle East respiratory syndrome (MERS), ZIKA, Dengue, hMPV(Human metapneumovirus), and RNMT (RNA Guanine-7 Methyltransferase). Ascan be seen, 2MR04 inhibits several MTase (SARS-nsp14, Dengue & Zikavirus MTases as well as hMPV MTase) in contrast to the RNMT (RNAGuanine-7 Methyltransferase), which is not inhibited. In addition, itwas observed that MR064 and HP-105 also inhibited Dengue virus NS5MTase.

Additional testing was conducted at 50 μM on other mononegavirus MTases(in triplicate):

-   1) human N7 MTase (hN7 or RNMT): 20 nM-   2) hMPV MTase: 1-   3) RSV MTase: 4-   4) SUDV MTase: 4

FIG. 7 shows that compound 2MR04 was effective in strongly inhibitingthe MTase activity of hMPV and RSV (Respiratory syncytial virus),viruses belonging to the family Pneumoviridae. The IC₅₀ values for 2MR04was determined and shown in FIG. 8 wherein the IC₅₀ for the hN7 MTasewas about 49 μM, about 31 μM on hMPV, and about 18 μM for RSV.

FIGS. 9 and 10 show the results for determining the IC₅₀ for the ZikaNS5-MTase and DV NS5-MTase using three compounds, that being, MR064,HP105 and 2MR04. It was found that 2MR04 strongly inhibits both the ZikaNS5-MTase and DV NS5-MTase with an IC₅₀ of about 22 μM. Also in FIG. 15,it is shown in another testing that 2 MR04 inhibited even stronger, thatbeing, with an IC50 of about 17 μM.

The fleximer 2MR04 was also tested for MERS and SARS-Cov. In vitroenzymatic assays were carried out in polymerase assay buffer (20 mMHepes pH 7.5; 10 mM KCl; 10 mM DTT; 4 mM MgCl2) in case ofMERS-Coronavirus and buffer (20 mM Tris pH 8; 10 mM KCl; 1 mM DTT; 2 mMMgCl₂) in case of SARS-Coronavirus. For the reaction, 1 μM of MERSpolymerase complex (12HC/7HC8) or 0.6 μM of SARS polymerase complex(12HC/7HC8) were mixed with 0.5 μM of a radiolabeled primer annealed toa template substrate and assembled at 30° C. for 30 min.

The primer extension was started by the addition of either 50 μM of eachpotential inhibitor or 50 μM of each inhibitor plus 20 μM of a NTPs mix.After incubation at 30° C., aliquots of reactions were quenched atvarious time points by the addition of an equal volume of loading buffer(formamide with 10 mM EDTA). Reaction products were loaded onto 20%polyacrylamide/7M urea gels, visualized using a Phosphorlmager (Fuji),and quantified using ImageQuant Software (Fuji).

As noted in FIG. 11, the inhibitor 2MR04 is as effective as the 3′ dGTPinhibitor (known as an N-pocket inhibitor) along with 2′-O-methyl GTPwhich is able to act as a chain terminator and inhibit RNA synthesis inthe testing for MERS inhibitors. The percentage of product formed isgreatly reduced relative to the control of 20 μM NTPs. In contrast. theresults for the inhibitor 2MR04 were found to be comparable to the otherinhibitors as shown in FIG. 12.

FIG. 13 shows blots of products formed using the MERS inhibitors and itis evident that the inhibitor 2MR04 (alone or with NTPs) was aseffective, not more effective, in reduction of oligonucleotidesynthesis. Likewise FIG. 14 shows that using the SARS inhibitors thatthe inhibitor 2MR04 (alone or with NTPs) was as effective in reductionof oligonucleotide synthesis.

REFERENCES

The contents of the references cited herein are incorporated byreference herein for all purposes.

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1. A method of preventing, treating and/or reducing the effects of aFilovirus, flavivirus or coronavirus in a subject in need of suchtreatment, the method comprising administering to the subject atherapeutically effective amount of an acyclic fleximer nucleosideanalogue selected from the group consisting of:

and a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.
 2. The method according to claim 1, wherein the acyclicfleximer nucleoside analogue is selected from the group consisting of:

or a pnarmaceuucaiiy acceptable salt, isomer, nyurate, prourug orsolvate mereof.
 3. The method of claim 2, wherein the acyclic fleximernucleoside analogue is structure 9 and the coronavirus is severe acuterespiratory syndrome (SARS) or Middle East respiratory syndrome (MERS).4. The method of claim 2, wherein the acyclic fleximer nucleosideanalogue is structure 9 and the Flavivirus is Dengue, Zika or West Nile.5. The method of claim 2, wherein the acyclic fleximer nucleosideanalogue is structure 3 and the Filovirus is Ebola, Sudan or andMarburg.
 6. The method of claim 1, wherein the acyclic fleximernucleoside analogue is in a composition further comprising apharmaceutically acceptable carrier.
 7. The method of claim 6, whereinthe acyclic fleximer nucleoside analogue is in a composition furthercomprising an additional antiviral agent.
 8. The method of claim 1,wherein the therapeutically effective amount of the acyclic fleximernucleoside analogue is from 0.05 to 50 mg per kilogram body weight ofthe subject per day.
 9. A method of binding to natural and/or mutatedpolymerases of a Filovirus, flavivirus or coronavirus in a subject inneed of such treatment, the method comprising administering to thesubject a therapeutically effective amount of an acyclic fleximernucleoside analogue selected from the group consisting of:

and a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.
 10. (canceled)
 11. An acyclic fleximer nucleosideanalogue having antiviral activity selected from the followingcompounds:

and a pharmaceutically acceptable salt, isomer, hydrate, prodrug orsolvate thereof.
 12. The acyclic fleximer nucleoside analogue accordingto claim 11, for use as a medicine or antiviral medicine with antiviralactivity that inhibits or reduces the effects of a Filovirus, flavivirusor coronavirus in a subject.
 13. The acyclic fleximer nucleosideanalogue according to claim 11, in a composition further comprising apharmaceutically acceptable carrier.
 14. The acyclic fleximer nucleosideanalogue according to claim 13, in a composition further comprising anadditional antiviral agent.